Vaccine compositions and method for enhancing an immune response

ABSTRACT

The invention relates to a vaccine which comprises an antigen and an immune response augmenting agent. The immune response augmenting agent is capable of enhancing T cell lymphokine production. Suitable immune response augmenting agents include, but are not limited to, dehydroepiandrosterone (DHEA) and DHEA-derivatives. Examples of DHEA derivatives include DHEA-sulfate (DHEA-S), 16α-bromo-DHEA, 7-oxo-DHEA, 16α-bromo-DHEA-S and 7-oxo-DHEA-S. 
     The invention also relates to a method for enhancing a vaccine-induced humoral immune response which comprises administering a vaccine which comprises an antigen and an immunomodulator. The immunomodulator may be an immune response augmenting agent, a lymphoid organ modifying agent or a mixture of the immune response augmenting agent and lymphoid organ modifying agent. Suitable lymphoid organ modifying agents include, but are not limited to, 1,25-dihydroxy Vitamin D 3 , biologically active Vitamin D 3  derivatives which are capable of activating the intracellular Vitamin D 3  receptor, all trans-retinoic acid, retinoic acid derivatives, retinol, retinol derivatives and glucocorticoid. Alternatively, the method for enhancing a vaccine-induced humoral immune response comprises separately administering the immunomodulator and a vaccine containing an antigen.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a continuation-in-part of U.S. application Ser. No.08/013,972, filed Feb. 4, 1993 abandoned, and of U.S. application Ser.No. 07/779,499, filed Oct. 18, 1991, abandoned, which in turn is acontinuation-in-part of U.S. application Ser. No. 07/412,270, filed Sep.25, 1989, abandoned.

TECHNICAL FIELD

The invention relates to vaccine compositions and methods of vaccinationwhich provide for higher antibody titres in the vaccinated individuals.More specifically, the invention relates to vaccine compositionscontaining immune response augmenting agents, such asdehydroepiandrosterone (DHEA) and DHEA derivatives as immunomodulatorsin the vaccine compositions. The invention further relates to methodsfor enhancing a vaccine-induced humoral immune response.

BACKGROUND OF THE INVENTION

It is known that lymphocytes exported from the thymus undergo a seriesof differentiation events which confer upon them the capacity torecognize and respond to specific peptide antigens presentedappropriately in the context of self major histocompatibility complex(MHC) molecules. Mechanistically, thymic maturation is a complex processwhich includes an irreversible rearrangement of T cell receptor genes,the cell surface expression of these gene products as disulfide-linkedheterodimers, positive and negative selection processes to provideappropriate restriction and avoidance of self-reactivity, and thesynthesis and expression of CD4 or CD8 as accessory adhesion molecules.Microenvironmental influences within the thymus play an essential rolein the fidelity of this process.

Subsequent to leaving the thymic microenvironment, mature T lymphocytesgain access to the recirculating T cell pool where they move freely viathe blood between mucosal and nonmucosal lymphoid compartments in themammalian host (Hamann et al. (1989). Immunol, Rev. 108: 19).T-lymphocyte expression of lymphoid tissue-specific homing receptors,which are complementary for vascular addressins on high endothelialvenules present in Peyer's patches and peripheral lymph nodes, provide abiochemical means for selectivity to this recirculation process (id.).Non-activated lymphocytes can move freely between mucosal and nonmucosallymphoid tissues due to the presence of both types of homing receptorson their plasma membranes (Pals et al. (1989). Immunol, Rev, 108: 111).Effector lymphocytes, and antigen-activated immunoblasts which arestimulated in a particular site in the body, however, exhibit a far moreselective migratory behavior. These cells move primarily to tissuesoriginally involved in antigen exposure and cellular activation (Hamannet al., supra; Pals et al., supra).

An immune response is initiated following T cell recognition of antigenpeptides in the context of self MHC molecules, and generally takes placein one of the host's secondary lymphoid compartments. Cellularactivation is triggered by the binding of antigen to the T cell receptor(TCR), forming an antigen/TCR complex which transduces theantigen-specific extracellular stimulation across the plasma membrane,and generates intracellular signals which include the activation ofprotein kinase C and the increases in intracellular calcium. Whilesignal transduction can lead to T cell unresponsiveness, positive signaltransduction events trigger a series of additional biochemicalprocesses. One consequence of this activation is the stimulatedproduction of a number of biologically active molecules, which arecollectively termed lymphokines. (See, Alcover et al. (1987). Immunol.Rev, 95: 5; Gelfand et al. (1987). Immunol. Rev. 95: 59).

Vaccines are preparations of antigenic material for administration toinduce in the recipient an immunity to infection or intoxication by agiven infecting agent. Vaccines may be prepared from viruses,rickettsiae, bacteria, protozoa and metazoa. Vaccines may be sterilesuspensions of the killed organisms, of toxoids or other antigenicmaterial derived from the organisms or recombinant sources, which can beadministered by injection. Vaccines may be either simple vaccinesprepared from one species of organism or a variety of organisms, or theymay be mixed vaccines containing two or more simple vaccines. They areprepared in such a manner as not to destroy the antigenic material,although the methods of preparation vary, depending on the vaccine.

Vaccine adjuvants consist of agents that are included in the formulationthat are used to enhance the ability of the antigenic material in avaccine to induce the desired immune response, and with many poorlyantigenic materials the success of vaccination depends on the presenceof a suitable adjuvant in the vaccine. The adjuvant is sometimesconveniently incorporated in the vaccine before the latter isdistributed into containers, although it may be provided in a separatecontainer for mixing with the antigenic material when the vaccine isrequired for use in immunizing the recipient.

U.S. Pat. No. 4,698,221 discloses a vaccine which contains (a) anantigen, (b) a fat-soluble vitamin, such as Vitamin A, Vitamin D and/orVitamin E, (c) a zinc compound, and (d) a selenium compound.

DHEA is a steroid hormone that has been extensively studied for manyyears. It has been reported to be involved in a wide variety ofphysiologic, immunologic, and pathologic conditions (for reviews, seeRegelson et al. (1988). Ann. N.Y. Acad. Sci. 521: 260; Gordon et al.(1986). Adv. Enzyme Reg. 26: 355-382). Most endocrinologists believethat the primary function of DHEA is to serve as a precursor for thesynthesis of testosterone and the estrogens by the gonads. Prior to itsrelease into the bloodstream, the vast majority of newly synthesizedDHEA becomes sulfated. The conjugated steroid DHEA-S is a secretoryproduct of the adrenal gland in humans and certain primates. DHEA-Srepresents the major steroid hormone in the circulation of humans, andis converted to DHEA by the enzymatic activity of asteroid sulfatase.

Therapeutic uses for DHEA and certain analogs have been reported fordiabetes, dry skin, ocular hypertension, obesity, and retrovitalinfections. Illustrative of these reports are the disclosures of U.S.Pat. No. 4,395,408, U.S. Pat. No. 4,518,595, U.S. Pat. No. 4,542,129,U.S. Pat. No. 4,617,299, U.S. Pat. No. 4,628,052, U.S. Pat. No.4,666,898, published European Patent Application No. 0 133 995 A2, andpublished United Kingdom Patent Application No. GB 2 204 237 A.

SUMMARY OF THE INVENTION

The invention relates to a vaccine which comprises an antigen and animmune response augmenting agent. The immune response augmenting agentis capable of enhancing T cell lymphokine production. Suitable immuneresponse augmenting agents include, but are not limited to, DHEA andDHEA-derivatives. Examples of DHEA derivatives include DHEA-sulfate(DHEA-S), 16α-bromo-DHEA, 7-oxo-DHEA, 16α-bromo-DHEA-S and 7-oxo-DHEA-S.

The invention also relates to a method for enhancing a vaccine-inducedhumoral immune response which comprises administering a vaccine whichcomprise's an antigen and an immunomodulator. The immunomodulator may bean immune response augmenting agent, a lymphoid organ modifying agent ora mixture of the immune response augmenting agent and lymphoid organmodifying agent. Suitable lymphoid organ modifying agents include, butare not limited to, 1,25-dihydroxy Vitamin D₃, biologically activeVitamin D₃ derivatives which are capable of activating the intracellularVitamin D₃ receptor, all trans-retinoic acid, retinoic acid derivatives,retinol, retinol derivatives and glucocorticoid. Alternatively, themethod for enhancing a vaccine-induced humoral immune response comprisesseparately administering the immunomodulator and a vaccine containing anantigen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the effect of DHEA and 1,25(OH₂)D₃administered in vivo on lymphokine production by activated splenocytes.

FIG. 2A is a graph showing the result of DHEA-S supplementation on thecapacity of T cells from aged mice to produce a IL-2.

FIG. 2B is a graph showing the result of DHEA-S supplementation on thecapacity of T cells from aged mice to produce IL-4.

FIG. 2C is a graph showing the result of DHEA-S supplementation on thecapacity of T cells from aged mice to produce IL-5.

FIG. 2D is a graph showing the result of DHEA-S supplementation on thecapacity of T cells from aged mice to produce γ-IFN.

FIG. 2E is a graph showing the result of DHEA-S supplementation on thecapacity of T cells from aged mice to produce IL-3.

FIG. 2F is a graph showing the result of DHEA-S supplementation on thecapacity of T cells from aged mice to produce GM-SCF.

FIG. 2G is a graph showing the results of chronic DHEA-S supplementationon the humoral responsiveness of aged mice to an ovalbumin (OVA)challenge. The responsiveness is shown for mature adult mice (□), agedmice (∘), and aged mice with oral DHEA-S ().

FIG. 3A is a graph showing the result of administration of a bolus ofDHEA-S to aged mice on the ability of splenocytes from the treated miceto have restored IL-2 production.

FIG. 3B is a graph showing the results of administration of a bolus ofDHEA-S to aged mice on the ability of splenocytes from the treated miceto have restored IL-4 production.

FIG. 3C is a graph showing the results of administration of a bolus ofDHEA-S to aged mice on the ability of splenocytes from the treated miceto have restored IL-5 production.

FIG. 3D is a graph showing the results of administration of a bolus ofDHEA-S to aged mice on the ability of splenocytes from the treated miceto have restored γ-IFN production.

FIG. 3E is a graph showing the results of administration of a bolus ofDHEA-S to aged mice on the ability of splenocytes from the treated miceto have restored IL-3 production.

FIG. 3F is a graph showing the results of administration of a bolus ofDHEA-S to aged mice on the ability of splenocytes from the treated miceto have restored GM-CSF production.

FIG. 3G is a graph showing the results of administration of a bolus ofDHEA-S to aged mice on the production of anti-ovalbumin antibodies bythe treated mice. The antibody production is shown for mature adult mice(□), aged mice (∘), and aged mice with DHEA-S injection ().

FIG. 4 is a graph showing the effect of topical DHEA application to agedmice on the production of anti-ovalbumin antibodies by the treated mice.The antibody production is shown for mature adult mice (□), aged mice(∘), aged mice with DHEA administration at the same site as the antigen(), and aged mice with DHEA administration at the opposite site of theantigen (▴).

FIG. 5A is a graph showing the effect of DHEA treatment in vivo on theproduction of IL-2 by activated splenocytes from thermally injured andcontrol mice.

FIG. 5B is a graph showing the effect of DHEA treatment in vivo on theproduction of γ-IFN by activated splenocytes from thermally injured andcontrol mice.

FIG. 5C is a graph showing the effect of DHEA treatment in vivo on theproduction of IL-4 by activated splenocytes from thermally injured andcontrol mice.

FIG. 5D is a graph showing the effect of DHEA treatment in vivo on theproduction of IL-5 by activated splenocytes from thermally injured andcontrol mice.

FIG. 5E is a graph showing the effect of DHEA treatment in vivo on theproduction of IL-3 by activated splenocytes from thermally injured andcontrol mice.

FIG. 5F is a graph showing the effect of DHEA treatment in vivo on theproduction of GM-CSF by activated splenocytes from thermally injured andcontrol mice.

FIG. 6 is a graph showing the effect of DHEA treatment in vivo oncontact hypersensitivity responses of thermally injured and controlmice.

FIG. 7 is a graph showing the effect of DHEA treatment on resistance toL. monocytogenes in control and thermally injured C3H mice.

FIG. 8 is a graph showing the effect of topically applied DHEA,16α-bromo-DHEA and 16α-chloro-DHEA on the anti-ovalbumin antibodyresponse in aged mice. The antibody response is shown for young mice(□), old mice (♦), old mice with DHEA (), old mice with 16α-bromo-DHEA(▴), and old mice with 16α-chloro-DHEA (▪).

FIG. 9 shows the effect of topical administration of DHEA on vaccinationof aged mice with recombinant Hepatitis B Surface Antigen (rHBSAg). Theprimary and secondary antibody responses are shown for aged mice withoutDHEA treatment (▪), aged mice with DHEA treatment at time of primaryimmunization (∘), and aged mice with DHEA treatment at time of secondaryimmunization (★).

FIG. 10A shows that primary and secondary antibody responses in agedmice following vaccination with rHBSAg is enhanced by topically appliedDHEA. The antibody responses are shown for mature mice without DHEAtreatment (▪), mature mice with DHEA treatment (), aged mice withoutDHEA treatment (▴), and aged mice with DHEA treatment (♦).

FIG. 10B shows that primary and secondary antibody responses in agedmice following vaccination with rHBSAg is enhanced by incorporating DHEAas a component of the vaccine. The antibody responses are shown formature mice without DHEA treatment (▪), mature mice with DHEA treatment(), aged mice without DHEA treatment (▴), and aged mice with DHEAtreatment (♦).

FIG. 11 shows that the antibody response in aged mice, followingvaccination with rHBSAg, is enhanced by treatment with DHEA or DHEA-S.The antibody responses are shown for aged mice without treatment (▪),aged mice with topical DHEA treatment (), aged mice with DHEAincorporated in the vaccine (▴), and aged mice with DHEA-S incorporatedin the vaccine (♦).

FIG. 12A shows that DHEA administration enhances the efficiency ofimmunization with suboptimal doses of rHBSAg. The antibody responses areshown for mice without DHEA treatment (▪) and with DHEA treatment (),for vaccination with 0.5 μg rHBSAg/mouse.

FIG. 12B shows that DHEA administration enhances the efficiency ofimmunization with suboptimal doses of rHBSAg. The antibody responses areshown for mice without DHEA treatment (▪) and with DHEA treatment (),for vaccination with 0.1 μg rHBSAg/mouse.

FIGS. 13A and 13B show that serum (systemic) antibody response in maturemice following vaccination with Influenza-A Beijing strain issynergistically enhanced by treatment with DHEA and 1,25(OH)₂ D₃.Antibody responses at Day 28 are shown for mice without treatment (▪),mice treated with 2 μg DHEA in vaccine (), mice treated with 0.1 μg1,25(OH)₂ D₃ in vaccine (▴), and mice treated with 2 μg DHEA and 0.1 μg1,25(OH)2D₃ in vaccine (♦).

FIGS. 13C and 13D shows that mucosal antibody response in mature micefollowing vaccination with Influenza-A Beijing strain is synergisticallyenhanced by treatment with DHEA and 1,25(OH)₂ D₃. Antibody responses atDay 28 are shown for mice without treatment (▪), mice treated with 2 μgDHEA in vaccine (), mice treated with 0.1 μg 1,25(OH)₂ D₃ in vaccine(▴), and mice treated with 2 μg DHEA and 0.1 μg 1,25(OH)₂ D₃ in vaccine(♦).

FIGS. 14A and 14B shows that serum (systemic) antibody response inmature mice following vaccination with rHBSAg is synergisticallyenhanced by treatment with DHEA and 1,25(OH)₂ D₃. Antibody responses atDay 21 are shown for non-immunized mice (★), mice without treatment (),mice treated with 2 μg DHEA in vaccine (♦), mice treated with 0.1 μg1,25(OH)₂ D₃ in vaccine (▴), and mice treated with 2 μg DHEA and 0.1 μg1,25(OH)₂ D₃ in vaccine (▪).

FIGS. 14C and 14D shows that mucosal antibody response in mature micefollowing vaccination with rHBSAg is synergistically enhanced bytreatment with DHEA and 1,25(OH)₂ D₃. Antibody responses at Day 21 areshown for non-immunized mice (★), mice without treatment (), micetreated with 2 μg DHEA in vaccine (♦), mice treated with 0.1 μg1,25(OH)₂ D₃ in vaccine (▴), and mice treated with 2 μg DHEA and 0.1 μg1,25(OH)₂ D₃ in vaccine (▪).

FIGS. 15A and 15B shows that serum (systemic) antibody response inmature mice following vaccination with rHBSAg is synergisticallyenhanced by treatment with DHEA and all trans-retinoic acid. Antibodyresponses at Day 21 are shown for non-immunized mice (▪), mice withouttreatment (), mice treated with. 5.0 μg all trans-retinoic acid invaccine (▴), and mice treated with 2 μg DHEA and 5.0 μg alltrans-retinoic acid in vaccine (♦).

FIGS. 15C and 15D shows that mucosal antibody response in mature micefollowing vaccination with rHBSAg is synergistically enhanced bytreatment with DHEA and all trans-retinoic acid. Antibody responses atDay 21 are shown for non-immunized mice (▪), mice without treatment (),mice treated with 5.0 μg all trans-retinoic acid in vaccine (▴), andmice treated with 2 μg DHEA and 5.0 μg all trans-retinoic acid invaccine (♦).

DETAILED DESCRIPTION OF THE INVENTION

The most important function of the immune system is to provide its hostwith protection against diseases. To carry out these tasks, a large anddiverse array of effector mechanisms have evolved, the majority of whichexhibit antigen specificity. Each individual effector mechanismpossesses a degree of uniqueness with respect to its ability toinfluence the rate of progression, to detoxify, or to promote theelimination of microbial pathogens or tumor cells. Such a diversity inavailable mechanisms is absolutely essential, since no single effectorresponse can effectively deal with all forms of pathogenic insults.Furthermore, to protect normal function of the various non-lymphoidorgan systems and tissues of the body requires careful selection,activation, and compartmentalization of the most appropriate types ofimmune effector mechanisms. Equally important is the simultaneouscapacity to down-regulate the development of other types of responses.Immunologic effector responses must, therefore, be both effective andpractical, and at the same time be appropriately regulated anatomicallyto reduce the risk of pathologic consequences.

The non-lymphoid tissues and organs of the body, which work collectivelyto sustain the life of the host, must also be capable of providingregulatory information to cells of the immune system. This information,mediated through the activities of inflammation-induced tissuecytokines, prostaglandins and other types of biological responsemodifiers, becomes integrated into the complex equation to control themechanisms which regulate effector response selection.

T cells, through their capacity to produce a number of lymphokines inresponse to activation, play a central role in guiding the developmentof immune effector responses. Mechanisms which operate to control thesynthesis and secretion of these pleiotropic biologic responsemodifiers, therefore, directly influence the quantitative andqualitative nature of immunity. The lymphokines and cytokines provideimportant information, not only to cells of the immune system, but alsoto cells of the other tissue and organ systems.. For this information tobe meaningful, it is essential that lymphokine production remainstightly controlled at the levels of both cellular source and duration.Autocrine and paracrine effects by lymphokines and cytokines should bethe norm, since only a few species are capable of working effectivelywhen provided via endocrine routes. These essential anatomicrestrictions, therefore, cannot be adequately provided by bolusinjection of recombinant lymphokines and/or cytokines, and may explainthe limited success associated with this form of therapy.

The vast majority of the T cells in the peripheral circulation are knownto reside within the recirculating T cell pool. These cells continuouslyenter and exit secondary lymphoid organs throughout the body,maintaining residence within any particular site for only finite periodsof time. Over the lifespan of any individual mature T cell, therefore,it has probably taken up temporary residence in most of a host'ssecondary lymphoid organs. T cell recirculation provides the immunesystem with a means for clonally-restricted T cells to provide a levelof surveillance over all of the tissue and organ systems.

It is universally accepted that most T cells acquire their specificityfor antigen, and for a self-MHC-restricting element, during processeswhich occur at the time of their ontogeny within the thymus. However,the extent to which intrathymic maturation confers genetic restrictionsupon individual T cells that regulate their potential for immunologicinvolvement has not been delineated.

A general concept which explains the results in the Examples, but whichis not intended to limit the invention, is that the genetic programs ofresting recirculating T cells are continuously being altered byextrinsic environmental influences. The steroid hormones, eitherpresented systemically in their active forms (e.g., asglucocorticosteroids (GCS)), or provided to T cells only within discretemicroenvironments as a consequence of end-organ metabolism (e.g., asDHEA, DHT or 1,25(OH)₂ D₃), perform important roles in this process. Thebasal regulation of the immune system at the level of the T cellrequires the continual presence of the needed substrates (prohormones).The anatomic compartmentalization of functional potential for T cells,therefore, would be dependent on the cellular source of the steroidmetabolizing enzymes able to convert the steroid hormone substrates totheir bioactive species. It has been shown that macrophages can containeach of these enzymes.

More specifically, DHEA-S can be naturally converted to DHEA in theperipheral lymph nodes of animals with normal immune function. The DHEAproduced then influences the T lymphocytes within the lymph node andexerts controlling influences on their ability to respond whenactivated. This provides a means to regulate the potential of T cells byfluctuating the degree to which a particular steroid hormone existswithin a particular tissue. Old individuals and/or stressed individuals,including humans, have a reduced capacity to produce DHEA-S, resultingin altered T-cell responsiveness. The invention in its variousembodiments restores the metabolite produced from DHEA-S in the anatomiccompartment in which appropriate T-cell responsiveness is required fornormal immune responses to T-cell-dependent antigens.

The invention relates to a vaccine which comprises an antigen and animmune response augmenting agent. The immune response augmenting agentis capable of enhancing T cell lymphokine production. Suitable immuneresponse augmenting agents include, but are not limited to, DHEA andDHEA-derivatives. Examples of DHEA derivatives include DHEA-sulfate(DHEA-S), 16α-bromo-DHEA, 7-oxo-DHEA, 16α-bromo-DHEA-S and 7-oxo-DHEA-S.

The invention also relates to a method for enhancing a vaccine-inducedhumoral immune response which comprises administering a vaccine whichcomprises an antigen and an immunomodulator. The immunomodulator may bean immune response augmenting agent, a lymphoid organ modifying agent ora mixture of the immune response augmenting agent and lymphoid organmodifying agent. Suitable lymphoid organ modifying agents include, butare not limited to, 1,25-dihydroxy Vitamin D₃, biologically activeVitamin D₃ derivatives which are capable of activating the intracellularVitamin D₃ receptor, all trans-retinoic acid, retinoic acid derivatives,retinol, retinol derivatives and glucocorticoid. Alternatively, themethod for enhancing a vaccine-induced humoral immune response comprisesseparately administering the immunomodulator and a vaccine containing anantigen.

As used herein, the term "immunomodulator" refers to an agent which isable to modulate an immune response. An example of such modulation is anenhancement of antibody production.

The term "individual" refers to a vertebrate and preferably to a memberof a species which naturally produces DHEA and DHEA-S and possessesDHEA-S sulfatase activity, and includes, but is not limited to domesticanimals, sports animals and primates, including humans.

The term "effective amount" of an immunomodulator refers to an amount ofan immunomodulator sufficient to enhance a vaccine-induced humoralimmune response. An effective amount of an immunomodulator, if injected,can be in the range of about 0.1-1,000 μg, preferably 5-500 μg, for ahuman subject, or in the range of about 0.01-10.0 μg/Kg body weight ofthe subject animal. This amount may vary to some degree depending on themode of administration, but will be in the same general range. If morethan one immunomodulator is used, each one may be present in theseamounts or the total amount may fall within this range.

An effective amount of an antigen may be an amount capable of elicitinga demonstrable humoral immune response in the absence of animmunomodulator. For many antigens, this is in the range of about 5-100μg for a human subject. Since the vaccines of the invention utilize animmunomodulator which enhances the humoral immune response, it may bepossible to utilize less antigen, e.g., about 1-5 μg for a humansubject.

The exact effective amount necessary will vary from subject to subject,depending on the species, age and general condition of the subject, theseverity of the condition being treated, the mode of administration,etc. Thus, it is not possible to specify an exact effective amount.However, the appropriate effective amount may be determined by one ofordinary skill in the art using only routine experimentation or priorknowledge in the vaccine art.

"Treatment" refers to the administration to an individual of acomposition which yields a protective immune response, and includesprophylaxis and/or therapy.

An "antigen" refers to a molecule containing one or more epitopes thatwill stimulate a host's immune system to make a secretory, humoraland/or cellular antigen-specific response. The term is also usedinterchangeably with "immunogen."

The specific antigen can be a protein, a polysaccharide, alipopolysaccharide or a lipopeptide; or it can be a combination of anyof these. Particularly, the specific antigen can include a nativeprotein or protein fragment, or a synthetic protein or protein fragmentor peptide; it can include glycoprotein, glycopeptide, lipoprotein,lipopeptide, nucleoprotein, nucleopeptide; it can include apeptide-peptide conjugate; or it can include a recombinant nucleic acidexpression product. Examples of antigens include, among others, thosethat are capable of eliciting an immune response against vital orbacterial hepatitis, influenza, diphtheria, tetanus, pertussis, measles,mumps, rubella, polio, pneumococcus, herpes, respiratory synctial virus,heamophilus influenza type b, chlamydia, varicellazoster virus orrabies.

An "immunological response" to a composition or vaccine comprised of anantigen is the development in the host of a cellular- and/orantibody-mediated immune response to the composition or vaccine ofinterest. Usually, such a response consists of the subject producingantibodies, B cells, helper T cells, suppressor T cells, and/orcytotoxic T cells directed specifically to an antigen or antigensincluded in the composition or vaccine of interest.

By "vaccine composition" or "vaccine" is meant an agent used tostimulate the immune system of an individual so that current harm isalleviated, or protection against future harm is provided.

"Immunization" refers to the process of inducing a continuing protectivelevel of antibody and/or cellular immune response which is directedagainst an antigen to which the organism has been previously exposed.

A "pharmacologic dose" is one which gives a desired physiologicaleffect.

"DHEA" or "DHEA-derivative", as used herein, refer to compounds havingthe formula ##STR1## wherein R¹ is ═O or OH;

R² is H or halogen;

R³ is H with a 5-6 double bond or ═O;

R⁴ is OR⁵ ;

R⁵ is H, SO₂ OM, PO₂ OM or a glucuronide group of the formula ##STR2##R⁶ and R⁷ may be the same or different and may be a straight or branchedC₁₋₁₄ alkyl.

"Lymphoid organ modifying agent," as used herein, means a biologicalresponse modifier that is capable, when administered to a vertebrateanimal in vivo at a peripheral site, of altering the microenvironment ofa peripheral lymphoid organ that drains from the administration site,such that activated lymphocytes and macrophages residing within thelymphoid organ exhibit a pattern of cytokines more typical of themicroenvironment of a lymphoid organ of the mucosal lymphoidcompartment. Particularly, a pattern of cytokines more typical of amucosal lymphoid organ is characterized by relatively enhancedproduction of one or more of active TGF-β, IL-4, IL-5, and IL-10, andrelatively decreased production (or at least no relatively enhancedproduction) of one or more of IL-2 and IFN-γ. In preferred embodiments,the lymphoid organ modifying agent includes a biological responsemodifier that can, or a combination of biological response modifiersthat together can, when administered to a peripheral lymph organ, resultin the lymphoid organ exhibiting a pattern of cytokines more typical ofa mucosal lymphoid organ. Lymphoid organ modifying agents are describedin U.S. application Ser. No. 08/013,972 and U.S. application Ser. No.08/123,844, U.S. Pat. No. 5,518,725, entitled "Vaccine Compositions andMethod for Induction of Mucosal Immune Response via SystemicVaccination," filed concurrently herewith, to latter of which isincorporated herein by reference. By way of example, it can be asubstance known to enhance or facilitate production of active TGF-β,such as, for example, all trans-retinoic acid, retinoic acid derivative,retinol or retinol derivatives, or it can be 1,25(OH)₂ D₃ or itsbiologically active derivatives, or possibly glucocorticoids.Biologically active Vitamin D₃ derivatives are those derivatives capableof activating the intracellular Vitamin D₃ receptor. Preferably, thelymphoid organ modifying agent is administered epicutaneously at aperipheral anatomical site (such as, for human subjects, for example,the arm or buttocks or leg); and the specific antigen is administered tothe same anatomical site, or to a site known to drain into the samelymphoid organ that receives drainage from the site of administration ofthe lymphoid organ modifying agent. In one mode of administration, thelymphoid organ modifying agent can be combined with the specific antigenand immune response augmenting agent for simultaneous administration atthe same site. The lymphoid organ modifying agent, the specific antigenand immune response augmenting agent, or all of them, can beadministered by injection.

An "immune response augmenting agent", as used herein, means an agentthat is capable, when administered to a vertebrate animal in vivo, ofrestoring T cell responsiveness to T cell dependent antigenscharacteristic of normal immune responses to such antigens. Immuneresponse augmenting agents are capable of enhancing T cell lymphokineproduction, particularly IL-2, IL-3, IFN-γ and GM-CSF. By way ofexample, the immune response augmenting agent can be a substance, suchas DHEA or DHEA-derivative, which enhances T cell lymphokine production.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,biochemistry, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, e.g., ANIMALCELL CULTURE (R. I. Freshney, Ed. 1986); IMMOBILIZED CELLS AND ENZYMES(IRL Press, 1986); the series, METHODS IN ENZYMOLOGY (Academic Press,Inc.), IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY (AcademicPress, London), Scopes, (1987); and HANDBOOK OF EXPERIMENTAL IMMUNOLOGY,Volumes I-IV, (D. M. Weir and C. C. Blackwell, Eds., 1986). All patents,patent applications and publications mentioned herein, both supra andinfra, are incorporated herein by reference.

The simultaneous enhancement or maximization of the production of morethan one T cell lymphokine may be achieved by exposing the T celllymphocyte to more than one immunomodulator prior to cellularactivation. The exposure to more than one immunomodulator can besimultaneous or sequential. The concentration of each of theimmunomodulators should be balanced to achieve the desired enhancingeffects on the vaccine-induced humoral immune response.

Evidence derived from experimental and clinical observations indicatesthat immunologic reactions elicited to either simple or complex antigensoften manifest as a balanced heterogenous blend of both cellular andhumoral components, with the fractional contribution of any individualtype of effector mechanism often dominating the overall response. Thislevel of heterogeneity is essential to the development of a protectiveimmune response. Alterations to this natural balance, whether caused bygenetic or physiologic changes associated with age or stress or trauma,can lead to a depressed capacity to elicit protective immune responses,and might also lead to immunologic responses having pathologicconsequences.

Pharmaceutical compositions made up of formulations comprised of theimmunomodulators and suitable for administration by subcutaneous,epicutaneous, topical, intramuscular or intradermal routes and the likemay be prepared by one of ordinary skill in the art. See, for example,Remington's Pharmaceutical Sciences, 17th Ed. (1985, Mack PublishingCo., Easton, Pa.). For example, the pharmaceutical compositioncontaining the immunomodulator may also contain a physiologicallyacceptable carrier that is non-toxic to the treated animal and iscompatible with the steroid. Suitable pharmaceutical carriers includeliquid carriers, such as normal saline and other non-toxic salts at ornear physiological concentrations.

The pharmaceutical composition containing the immunomodulator is used asa vaccine adjuvant to enhance a vaccine-induced humoral immune response.When the individuals are immunized with an immunizing agent,administration of the immunomodulator may be prior to, contemporaneouslywith, or after the vaccination. Typical methods of administering theimmunomodulator include mixing the immunomodulator with the immunizingagent (antigen) in a vaccine or topically applying the immunomodulatorto skin sites which drain into the same lymph nodes as the antigen ofthe vaccine. This latter method is preferably used with individuals whoare immunologically deficient due to low levels of DHEA-S and/or DHEAand in whom one wishes to augment the immune response, for example, theaged or neonates or individuals who are therapeuticallyimmunosuppressed.

One or more immunomodulators may be used to enhance the vaccine-inducedimmune response. They may be administered sequentially orcontemporaneously. It is preferred to administer them contemporaneouslyand in a single vehicle. It has been discovered that a synergisticenhancing effect is achieved when an immune response augmenting agent,as defined herein, is combined with a lymphoid organ modifying agent, asdefined herein, and used as the immunomodulator. It is preferred toinclude the two agents in the vaccine, although either or both may alsobe topically applied. In general, an effective amount of immunomodulatormay be about 0.1-1,000 μg, preferably 5-500 μg, for a human subject. Aneffective amount of an immune response augmenting agent may be about10-1,000 μg, preferably 20-200 μg, for a human subject if administeredby injection. If the immune response augmenting agent is administeredorally, the amount may range from about 10-100 mg/day, preferably 20-50mg/day, for a human subject, or in the range of about 0.5-5 mg/Kg/dayfor an animal subject. An effective amount of a lymphoid organ modifyingagent may be about 0.1-500 μg, preferably 0.5-250 μg, for a humansubject.

Described below are examples of the present invention which are providedonly for illustrative purposes, and not to limit the scope of thepresent invention. In light of the present disclosure, numerousembodiments within the scope of the claims will be apparent to those ofordinary skill in the art.

EXAMPLE 1 The Effect of DHEA and 1,25(OH)₂ D₃ in vivo on IL-2 and IL-4Production in vitro

C3H mice received implants of biodegradable DHEA or 1.25(OH)₂ D₃ pelletsdesigned to deliver steroid at a rate of 10.4 and 2.6 μg/hour,respectively. Three days after implantation, both the steroid treatedgroups of mice and a normal control group were immunized in the hindfootpads with 100 μg OVA in CFA. Ten days after immunization, thedraining lymph nodes and spleens from all groups were prepared forculture. Lymph node cells were stimulated with 100 μg OVA. Culturesupernatants were assayed for IL-2 and IL-4 activity after 24 hoursusing the HT-2 bioassay. The results are shown in FIG. 1. From theFigure, it is seen that DHEA administration caused an approximatelyfour-fold increase in IL-2 production, and no stimulation of IL-4production. In contrast, 1,25(OH)₂ D₃ administration caused anapproximately eight-fold increase in IL-4 production, but did notstimulate IL-2 production.

Similar alterations in the ability of antigen-activated T cells toproduce IL-2 and IL-4 were observed when the immune response augmentingagent was mixed with the immunizing antigen, or was topically applied toskin sites above the site of vaccination.

EXAMPLE 2 Preservation of Normal Potential to Produce T-cell Lymphokinesand Generate Humoral Immune Responses by Supplementation with DHEASulfate

Circulating levels of DHEA sulfate decline markedly with advancing agein humans and other mammals. As shown in U.S. application Ser. No.07/779,499, abandoned, direct treatment of T cells from aged or normalmurine donors with DHEA prior to activation in vitro augmented theircapacity to produce IL-2. In contrast, DHEA-S, the prohormone form ofDHEA found principally in the circulation, was shown to have no directeffect on T-cell production of this lymphokine. When DHEA-S wasadministered to normal mature adults in vivo, it enhanced the potentialfor IL-2 production by T cells isolated from lymphoid organs having thegreatest DHEA-S activity. The most active lymphoid organs are thosehaving anatomic positions downstream of nonmucosal tissues. This Exampledemonstrates that DHEA-S supplementation in vivo can influence theage-relates changes in lymphokine production and humoral immuneresponses.

Groups of adult BALB/c mice, between 35 and 39 weeks of age, wereseparated into two groups. One group was provided with 100 μg/ml DHEA-Sin their drinking water. The hormone was offered ad libitum to theseanimals. The other group was left untreated. Mice were maintained onoral DHEA-S supplementation until age 114 weeks, when they weresacrificed and their spleens individually analyzed for the capacity toproduce lymphokines following anti-CD3ε activation. The DHEA-S treatedand untreated mice were evaluated by comparing their responses to thelymphokine profile produced by similarly activated splenocytes frommature adult mice (13 weeks of age).

More specifically, splenocytes were prepared from the following groupsof BALB/c mice; 2 mature adult (13 weeks), 2 aged (114 weeks), and 2aged (114 weeks) receiving 100 μg/ml DHEA-S in their drinking water forthe previous 61 weeks. 1×10⁷ splenocytes were cultured under serum-freeconditions in triplicate and activated with 1 μg/ml CD3ε. Culturesupernatants were analyzed for the level of IL-2 by a quantitativebioassay, and for IL-4, IL-5, γ-IFN, IL-3 and GM-CSF by capture ELISA.

FIGS. 2A-2F are graphs is a graph showing the results of DHEA-Ssupplementation on the capacity of T cells to produce a variety oflymphokines. In FIGS. 2A-2F, bars represent the mean ±SD for the valueof each lymphokine presented. It is seen from FIGS. 2A-2F that DHEA-Ssupplementation, administered prior to the onset of age-induced declinein immunocompetence, is accompanied by the preservation of normallymphokine production and development of normal humoral immuneresponses. DHEA-S supplementation was not only able to preserve normallevels of IL-2, IL-3, and GM-CSF production by activated T cells, butwas also able to prevent age-related increase in γ-IFN, IL-4, and IL-5production seen in the cell supernatants from untreated aged donors. Theresults of this study demonstrate that a striking correlation existsbetween the age-related decline in endogenous DHEA production (plus itsmetabolites) and the age-associated alterations in T-cell production oflymphokines.

The effect of DHEA-S supplementation on T cell function was alsoperformed using BALB/c, C57BL/6 and C3H/HeN strains of mice. In eachtest of this experimental approach, lymphokine production by T cellsfrom the treated aged donors had been preserved.

In order to examine the effect of DHEA-S supplementation on the abilityof old animals to mount immunologic responses to challenge with foreignprotein antigens, the following procedure was used. Groups of 5 matureadult mice (13 weeks), 5 aged mice (114 weeks), and 5 aged mice (114weeks) provided with chronic DHEA-S supplementation (100 μg/ml DHEA-S intheir drinking water for the previous 61 weeks, initiated at 8 months ofage), were footpad-immunized with ovalbumin. The immunization was with100 μg ovalbumin in a 25 μl volume of Maalox, administered in the hindfootpads. All animals were bled on Days 0, 3, 5, 7, 10, and 14post-immunization, and individual serum samples analyzed for ovalbuminspecific antibody titres by quantitative ELISA, using ovalbumin forcapture and HRPO-coupled, goat anti-murine Ig detecting antibodies withspecificity for IgM and IgG subclasses. Each ELISA assay was controlledwith sera known to be positive or negative for anti-ovalbumin activity.The titre is the inverse of the antibody dilution equal to thehalf-maximal point on the titration curve. The results of the study,shown in the graph in FIG. 2G, demonstrate that old animals providedwith chronic DHEA-S supplementation remain fully capable of rapidlymounting a significant humoral immune response to ovalbuminimmunization, with kinetics, titres, and isotype profiles that arealmost identical to mature adult controls. As expected, the untreatedaged mice responded poorly to a similar antigen challenge, producingpredominantly IgM.

EXAMPLE 3 DHEA-S Administration to Aged Mice Can Reverse Age-AssociatedChanges in T-cell Lymphokine Production and Their Depressed HumoralImmune Responses to Protein Antigens

As shown in U.S. application Ser. No. 07/779,499, abandoned, a directexposure of lymphocytes from aged donors to DHEA in vitro immediatelyaltered the pattern of lymphokines produced following activation. Inaddition, it has been found that nonmucosal tissue-draining lymphoidorgans possess a far greater amount of DHEA sulfatase activity thanmucosal tissue-draining lymphoid organs. These findings led to thehypothesis that DHEA may be serving as an effector of positionalinformation for lymphocytes residing in certain lymphoid compartments.Any changes in immune function caused by the depressed production ofsubstrate DHEA-S might, therefore, be reversible if DHEA-S isreintroduced in situ. This was examined in the following studies.

Splenocytes were isolated from equal-sized groups of mature adult mice(25 weeks), aged mice (120 weeks), and aged mice (120 weeks) given asubcutaneous injection of DHEA-S (100 μg in 100 μl propylene glycol) 24hours previously. 1×10⁷ splenocytes were cultured under serum-freeconditions in triplicate and activated with 1 μg/ml anti-CD3ε.Twenty-four hours later, culture supernatants from individual cellcultures were analyzed for the level of In-2 by a quantitative bioassay,and for IL-4, IL-5, γ-IFN, IL-3 and GM-CSF by capture ELISA. Theresults, shown in FIGS. 3A-3F, demonstrate that acute replacementtherapy to aged mice with DHEA-S restores near-normal patterns of T-celllymphokines within one day of treatment. These results strongly showthat lymphoid cells from old animals exhibit no intrinsic defects.Rather, some of the best documented functional changes to the immunesystem which accompany aging are due to the reduced capacity to produceDHEA-S.

A representative study, showing that administration of a bolus of DHEA-Sto aged BALB/c mice restored the capacity of the mice to develop humoralresponses, is shown in FIG. 3B. In the study, groups of 5 mature adult(25 weeks), 5 aged (120 weeks), and 5 aged (120 weeks) mice receiving100 μg DHEA-S in 100 μl propylene glycol by subcutaneous injection theprevious 24 hours were immunized with 100 μg ovalbumin in a 25 μl volumeof Maalox, administered in the hind footpads. Sera from individual micewere collected on Days 0, 3, 5, 7, 10 and 14 following primaryimmunization. The titre of anti-ovalbumin antibody was assessed by ELISAusing ovalbumin for capture and HRPO-coupled, goat anti-murine Igdetecting antibodies with specificity for IgM and IgG subclasses. EachELISA assay was controlled with sera known to be positive or negativefor anti-ovalbumin activity.

The results in FIG. 3G show that old animals provided With DHEA-S only24 hours prior to immunization with a foreign protein antigen respondedeven better than normal mature adults in the production of antibody.

This method of reversing age-related decline in humoral responses hasbeen evaluated twice using BALB/c mice and once with C3H/HeN strains ofmice. Similar enhancements in antibody production were achieved in allgroups of DHEA-S treated, aged groups of mice.

The results described in U.S. application Ser. No. 07/779,499,abandoned, and discussed above demonstrate that some of theage-associated changes in immune function are extrinsic in cause, andare mediated by the loss in endogenous production of an essentialregulatory steroid prohormone.

EXAMPLE 4 Topical Application of DHEA to Aged Animals FacilitatesChanges in the Draining Lymph Node Microenvironment that are Conduciveto Successful Immunization

Groups of 5 mature adult (13 weeks) and 10 aged BALB/c mice (114 weeks)were used in the study. All of the aged BALB/c mice received a topicalapplication of 10 μg DHEA in 3.5 μl 95% ethanol to the right hindfootpad, 3 hours prior to immunization with 100 μg ovalbumin in a 25 μlvolume of Maalox. Five of the aged mice were immunized in the right hindfootpad (at a site identical to the steroid application), and the other5 immunized in the left hind footpad (site opposite to the steroidapplication). Sera from individual mice were collected on Days 0, 3, 5,7, 10 and 14 following primary immunization. The titre of anti-ovalbuminantibody was assessed by ELISA using ovalbumin for capture andHRPO-coupled, goat anti-murine Ig detecting antibodies with specificityfor IgM and IgG subclasses. Each ELISA assay was controlled with seraknown to be positive or negative for anti-ovalbumin activity.

The results, shown in FIG. 4, establish that a topical application ofDHEA, prior to immunization through the same skin site, provided theaged animals with the ability to generate completely normal humoralimmune responses. The untreated group of aged animals, and aged animalsprovided with topical DHEA on the footpads opposite the site ofimmunization, responded quite poorly to immunization, with minimalantibody being observed.

The results of reversing the age-related decline in humoral responseshas been repeated with BALB/c mice, and with C3H/HeN strain of mice. Theresults establish that the pronounced lymphoid organ-specific changes inthe types of lymphokines produced by T cells from aged animals giventopical DHEA, can be paralleled by an equally dramatic enhancement inability to generate potent humoral immune responses to challenge with aforeign antigen protein.

EXAMPLE 5 Treatment in vivo of Thermally-Injured Mice with DHEAPreserves Normal Immune Function

U.S. application Ser. No. 07/779,499, abandoned, demonstrates thatthermal injury to mice caused a depression in the capacity of activatedT-cells to secrete IL-2, IFN-γ, IL-3 and GM-CSF, and that thisdepression could be prevented with DHEA treatment. The following studyillustrates that direct administration of DHEA to mice shortly afterthermal injury influences their levels of immunocompetence.

Groups of 12 thermally-injured and 6 control BALB/c mice wereestablished as described above. After subjecting the mice to a 20% totalbody surface area (TBSA) scald burn, six of the thermally-injured micewere given a subcutaneous injection of 100 μg DHEA in a propylene glycolcarrier. All remaining animals received the carrier alone. Five dayslater, all surviving mice were sacrificed and their splenocytes wereindividually prepared for culture, and activated with anti-CD3ε toinduce lymphoine secretion. Culture supernatants were collected 24 hoursafter activation and evaluated for lymphokine content, as described inU.S. application Ser. No. 07/779,499, abandoned. The results of thestudy are presented in FIGS. 5A-5F, where the bars represent mean ±SDfor each value. As seen in FIGS. 5A-5F, DHEA directly influences IL-2,γ-IFN, IL-3, and GM-CSF production by T cells isolated fromthermally-injured mice. The administration of a single bolus injectionof DHEA (100 μg) 1 hour after thermal injury was sufficient to preservefor at least 5 days a normal capacity by their lymphocytes to produceIL-2, γ-IFN, IL-3, and GM-CSF following activation. No significantchanges from normal were observed in the levels of IL-4 and IL-5 made byactivated lymphoid cells from these animals.

The effect of DHEA treatment in vivo on the animals' development ofcellular immune responses was examined. Parallel groups ofthermally-injured and control mice were given either 100 μg DHEA inpropylene glycol carrier or the carrier alone 1 hour post-burn. Theseanimals were contact-sensitized 5 days later by administration of DNFBon the abdomen. Challenge doses of DNFB to the right footpads wereapplied 4 days later. The differences in thickness between the right(challenged) and the left (unchallenged) footpads were used toquantitate the contact hypersensitivity responses. The results are shownin FIG. 6; the bars represent mean ±SD for each group of mice. As shownin FIG. 6, the intensity of contact hypersensitivity responses elicitedby thermally-injured mice is markedly depressed, as compared tocontrols. Administration of DHEA to thermally-injured mice was found tocompletely preserve the ability of these animals to develop contacthypersensitivity responses of normal intensity.

These studies demonstrate that DHEA treatment post-burn is an effectivetherapy for preserving the capacity of T-lymphocytes fromthermally-injured animals to produce normal quantities of a number oflymphokines, especially those that are essential for development ofcellular immune responses. This finding is supported by thedemonstration in U.S. application Ser. No. 07/779,499, abandoned, thatDHEA-treated thermally-injured mice also retain their capacity todevelop normal contact hypersensitivity responses.

EXAMPLE 6 DHEA Treatment in vivo Promotes Resistance to Infection by L.monocytogenes in Thermally-Injured Mice

This study addresses the utility of DHEA therapy post-burn in preservingresistance to a bacterial infection. C3H/HeN strain mice are inherentlyresistant to infection by the gram-positive intracellular pathogen, L.monocytogenes. However, thermal injury results in increasedsusceptibility to this pathogen. Therefore, a switch from "resistant" toa more "susceptible" phenotype provides a model system to evaluate theeffect of DHEA on preserving the "resistant" phenotype inthermally-injured animals.

Normal (control) and thermally-injured mice were prepared as describedabove. Half of the thermally-injured mice received a single bolusinjection of 100 μg DHEA subcutaneously within 1 hour after thermalinjury. Three days later, all mice were infected with 2×10⁶ viable L.monocytogenes organisms, and 3 days after infection the mice weresacrificed and homogenates of individual spleens were prepared. Thenumber of colonies of L. monocytogenes per spleen were evaluated usingstandard methodology, and scored. The results are presented graphicallyin FIG. 7, where the bars represent means ±SEM for each treatment group.The results indicate that thermal injury enhances the susceptibility ofthe C3H strain mice to infection by L. monocytogenes. Of consequence,DHEA treatment of burned animals not only preserves the resistantphenotype, but surprisingly, the level of resistance to infection isactually enhanced by DHEA treatment over that observed in the controlgroup.

EXAMPLE 7 The Effect of 16α-bromo-DHEA and 16α-chloro-DHEA on theDepressed Humoral Responses of Aged Mice to Protein Antigens

The effectiveness of the cogeners of DHEA, 16α-bromo-DHEA and16α-chloro-DHEA on age-associated changes in the humoral immune responseis demonstrated in the following study. Groups of mature (young) andaged (old) mice were treated by topical administration of 10 μg of DHEA,16α-bromo-DHEA, or 16α-chloro-DHEA. Three hours subsequent to treatment,the treated and control animals were subjected to an ovalbumin (OVA)challenge as described in the Examples above, and the anti-ovalbuminantibody response was measured at Days 0, 3, 5, 7, 10, and 14 afterimmunization. The results, shown in FIG. 8, indicate that 16α-bromo-DHEAis as effective as DHEA in restoring humoral immune responsiveness. The16α-chloro-DHEA yielded a lower, but significant, effect on antibodyproduction.

EXAMPLE 8 Topical Administration of DHEA Enhances Antibody Productionwith Vaccines to Hepatitis B Surface Antigen

Groups of five mice [(C3H/HeN×C57BL/6)F1], 72 weeks of age, wereimmunized with 1.5 μg Hepatitis B surface antigen (HBSAg) in aluminumhydroxide (273 μg/ml; alum). Five of the mice were pre-treated with 10μg DHEA by topical administration 3 hours prior to immunization. Serafrom individual mice were collected during the primary response. Priorto a secondary immunization on Day 50, five of the aged control micewere treated with 10 μg DHEA by topical administration. All of the micewere then given a secondary immunization of 1.5 μg HBSAg in alum andsera were collected weekly. Specific response to the vaccination wasmeasured by quantitative ELISA. Purified rHBSAg diluted in 0.5MTris-HCl, pH 9.6, at a concentration of 2 μg/ml was dispensed into96-well plates. Following incubation for a minimum of two hours at roomtemperature, or overnight at 4° C., all plates were blocked withPBS-0.05% Tween 20/1.0% bovine serum albumin (BSA) for an additionaltwo-hour incubation at room temperature. Prior to adding the testsamples, the plates were washed free of blocking buffer using threewashes of distilled water and one wash with PBS/0.05% Tween 20.Individual samples were first diluted in PBS 0.05% Tween 20/1.0% BSA,and 100 μl was then dispensed into appropriate wells of theantigen-coated plates. Included on each plate was an Ig standard: aseries of two-fold dilutions of either purified IgG (all subclasses) orIgA (reference standards). The reference Ig were captured by goatanti-murine Ig which is known to bind all murine Ig isotypes. Theseplates were incubated at room temperature overnight, followed by 3× washin distilled water and one wash in PBS/0.05% Tween 20. The detectionantibody (HRP-lined goat anti-mouse Ig specific for IgG and IgA) wasdiluted in PBS/Tween/10% normal goat serum at a dilution recommended bythe manufacturer. After a final incubation and wash series, the ELISAwas developed using ABTS-substrate. O.D. readings were recorded at 405nM using a Vmax 96-well microtest plate spectrophotometer (MolecularDevices, Menlo Park, Calif.). A simple linear regression analysis of theIg titration generated a reference curve for calculating the amount ofspecific antibody contained in the test samples. These data are reportedas ng/ml ±SEM. The results are shown in FIG. 9, and confirm the findingsshown in Examples 2 and 3. FIG. 9 demonstrates that topicaladministration of DHEA provides an enhancement in the immune responsefor vaccination with HBSAg, as seen by serum anti-HBSAg antibodies.

Similar effects were seen with the topical administration of16-α-Br-DHEA.

EXAMPLE 9 Administration of DHEA Topically Prior to Vaccination or as aComponent of the Vaccine Enhances Antibody Production

Aged mice [(C3H×BL/6)F1], approximately 24 months old, were given atopical application of 10 μg DHEA three hours prior to vaccination witha recombinant Hepatitis B surface antigen (rHBSAg). Alternatively, 10 μgDHEA was incorporated into the vaccine/alum mixture prior toimmunization. Untreated aged mice, and untreated mature adult mice[C3H×BL/6)F1], 26 weeks of age, were administered the ethanol vehiclewithout DHEA. All of the test mice were vaccinated with rHbSAg (1.0 μg)in 25 μl alum (273 μg/ml) through the anatomic site of DHEA or ethanolapplication. Serum samples were collected from individual mice atmultiple times during the primary response. A secondary antigen exposurewas given to all mice 50 days after the primary immunization. Recallresponses were stimulated without any additional treatment with DHEA.Serum samples were then collected 5 and 10 days following secondaryantigen exposure. Individual serum samples were evaluated byquantitative ELISA as described above to determine the amount ofHBSAg-specific antibody. The mean and SEM antibody responses at eachinterval are shown in FIGS. 10A and 10B. The results show that the serumantibody response to rHBSAg was enhanced in aged mice when the mice weretreated prior to vaccination with topical DHEA (FIG. 10A) or weretreated with DHEA incorporated in the vaccine (FIG. 10B).

EXAMPLE 10 Administration of DHEA or DHEA-S Topically or as a Componentof the Vaccine Enhanced Antibody Production

Groups of sex- and age-matched mice [(C3H×C57 BL/6)F1], greater than 24months of age, were immunized subcutaneously with rHBSAg (1.0 μg in 25μl alum (273 μg/ml)). Animals were given a topical administration of 10μg DHEA three hours prior to vaccination. Alternatively, 10 μg DHEA orDHEA-S was incorporated into the vaccine/alum mixture prior toimmunization. Untreated aged mice were administered the ethanol vehiclewithout DHEA. Serum samples were collected from individual mice atmultiple times and were evaluated by quantitative ELISA as describedabove, to determine the amount of HbSAg-specific antibody. The meanantibody response is shown in FIG. 11. The results show that the serumantibody response was enhanced in aged mice when the mice were treatedprior to vaccination with topical DHEA () or were treated with DHEA (▴)or DHEA-S (♦) incorporated in the vaccine.

EXAMPLE 11 Administration of DHEA Enhances Immunization with SuboptimalDoses of rHBSAg

Groups of 8 mature adult CF-1 mice were immunized with either 0.5 μg or0.1 μg rHBSAg in 25 μl alum (273 μg/ml), subcutaneously in a single hindfootpad. Four mice in each group were pre-treated with either 10 μg DHEAin ethanol or the ethanol vehicle alone, topically applied to the samesite as the immunization. Serum samples from individual mice werecollected and analyzed for specific antibody, as described above. Thescattergram shows the responses in ng/ml of each mouse to the vaccine at28 days post-immunization. FIG. 12 shows that the antibody response torHBSAg was enhanced for the suboptimal doses of 0.5 μg rHBSAg (FIG. 12A)and of 0.1 μg rHBSAg (FIG. 12B).

EXAMPLE 12 Topical Administration of DHEA Enhances Serum AntibodyProduction in Elderly Mice Upon Vaccination

Examples 2 and 3 show that DHEA-S administration restored the ability ofaged mice to mount a humoral immune response against ovalbumin. Thisexample demonstrates that topical administration of DHEA prior tovaccination against several different antigens enhances serum antibodyproduction in elderly mice.

Aged mice [C3H/HeN], approximately 22-27 months of age, were given atopical application of 10 μg DHEA three hours prior to vaccination withDiphtheria toxoid (Dr, 1.0 μg), Tetanus toxoid (Tt, 1.0 μg), or aHemophilus Influenza Type b conjugate vaccine coupled to Dt (500 ng ofHib polysaccharide chemically coupled to 1.25 μg Dt) in standard alumadjuvant. Untreated aged mice and untreated mature adult mice [C3H/HeN],17-24 weeks of age, were administered the ethanol vehicle without DHEA.Serum samples were collected from individual mice at multiple timesduring the primary response. Individual serum samples were evaluated byquantitative ELISA as described above, using purified Dt (diluted in0.05M Tris-HCl (pH 9.6) at a concentration of 2.0 μg/ml), purified Tt(diluted in 0.05M Tris-HCl (pH 9.6) at a concentration of 2 μg/ml), orHib-meningococcal protein conjugate (diluted in 0.05M Tris-HCl (pH 9.6)at a concentration of 200 ng/ml polysaccharide). The mean primaryantibody response at Day 28 is shown in Table 1. The results show thatthe serum antibody responses to these antigens were enhanced in agedmice with topical administration of DHEA prior to vaccination. Similarresults are obtained when DHEA is incorporated in the vaccine.

                  TABLE 1                                                         ______________________________________                                        Serum Antibody Production                                                                  Primary Antibody Response (μg/ml)                                                               DHEA-                                                      Mature             treated                                     Immunogen      Adult      Aged    Aged                                        ______________________________________                                        Diphtheria     1.65       0.43    5.75                                        Toxoid (1.0 μg)                                                            Tetanus        6.8        2.3     4.6                                         Toxoid (1.0 μg)                                                            Hemophilus Influenza                                                          Type b Conjugate                                                              Vaccine:                                                                      Hib Polysaccha-                                                                              13.3       6.4     16.2                                        ride (500 ng)                                                                 Diphtheria     5.8        3.5     7.8                                         Toxoid (1.25 μg)                                                           ______________________________________                                    

EXAMPLE 13 Administration of DHEA-S in Vaccine Enhances Serum AntibodyProduction in Elderly Mice

Examples 2 and 3 showed that DHEA-S administration restored the abilityof aged mice to mount a humoral immune response against ovalbumin.Example 12 demonstrated that topical administration of DHEA prior toimmunization enhanced the production of serum antibodies in elderlymice. This Example demonstrates that administration of DHEA-S in theantigen vehicle also enhances serum antibody production in elderly mice.

Aged mice [C3H/HeN], 22-27 months of age, were vaccinated withinactivated Influenza A Beijing strain (0.1 μg) in a standard alumadjuvant (25 μl; 273 μg alum/ml) which also contained 10 μg DHEA-S.Untreated aged mice and untreated mature adult mice [C3H/HeN], 17-24weeks of age, were administered the vaccine without DHEA-S. Serumsamples were collected from individual mice at multiple times during theprimary response. Individual serum samples were evaluated byquantitative ELISA as described above, using purified inactivatedInfluenza A (diluted in 0.05 M Tris-HCl (pH 9.6) at a concentration of0.1 μg/ml). The mean primary antibody response at Day 28 is shown inTable 2. The results show that the serum antibody response to thisantigen was enhanced in aged mice with incorporation of DHEA-S in thevaccine.

                  TABLE 2                                                         ______________________________________                                        Serum Antibody Production                                                               Primary Antibody Response (μg/ml)                                                                DHEA-                                                     Mature              treated                                       Immunogen   Adult       Aged    Aged                                          ______________________________________                                        Inactivated 4.7         0.16    4.3                                           Influenza A                                                                   Virus (0.1 μg)                                                             ______________________________________                                    

EXAMPLE 14 Administration of 1,25(OH)₂ D₃ With Vaccination to DiphtheriaToxoid Enhances Antibody Response

Groups of 3-5 age-matched C3H/HeN female mice were given primaryimmunization subcutaneously in the right footpad with 10 μg Diphtheriatoxoid (Dr; Connaught Laboratories) in alum (273 μg/ml). One group ofmice were administered 2μg 1,25(OH)₂ D₃ epicutaneously to the rightfootpad surface on Day 0. A second group of mice was similarlyadministered 1,25(OH)₂ D₃ on Day 5 after immunization. A third group ofmice (control) received an equal volume of the ethanol carrier. Afterweekly sampling of serum, mice were secondarily immunized subcutaneouslythrough an intrapelvic route with Dt and no additional exposure to1,25(OH)₂ D₃. Serum samples were collected and all primary and secondarysamples were then assayed individually using a Dt-specific, quantitativeELISA for IgG and IgA, using purified Dt diluted in 0.05 M Tris-HCl (pH9.6) at a concentration of 2.0 μg/ml. The purified Dt was dispensed into96-well plates. Following incubation for a minimum of two hours at roomtemperature, or overnight at 4° C., all plates were blocked withPBS-0.05% Tween 20/1.0% bovine serum albumin (BSA) for an additionaltwo-hour incubation at room temperature. Prior to adding the testsamples, the plates were washed free of blocking buffer using threewashes of distilled water and one wash with PBS/0.05% Tween 20.Individual samples were first diluted in PBS 0.05% Tween 20/1.0% BSA and100 μl was then dispensed into appropriate wells of the antigen-coatedplates. Included on each plate was an Ig standard: a series of two-folddilutions of either purified IgG (all subclasses) or IgA (referencestandards). The reference Ig were captured by goat anti-murine Ig whichis known to bind all murine Ig isotypes. These plates were incubated atroom temperature overnight, followed by 3× wash in distilled water andone wash in PBS/0.05% Tween 20. The detection antibody (HRP-lined goatanti-mouse Ig specific for IgG and IgA) was diluted in PBS/Tween/10%normal goat serum at a dilution recommended by the manufacturer. After afinal incubation and wash series, the ELISA was developed usingABTS-substrate. O.D. readings were recorded at 405 nM using a Vmax96-well microtest plate spectrophotometer (Molecular Devices, MenloPark, Calif.). A simple linear regression analysis of the Ig titrationgenerated a reference curve for calculating the amount of specificantibody contained in the test samples. These data are reported as ng/ml±SEM. It was found that mice which received 1,25(OH)₂ D₃ at Day 0 showeda slight enhancement in serum levels of IgG. Mice which received1,25(OH)₂ D₃ at Day 0 or Day 5 showed a substantial enhancement in serumlevels of IgA. Substantial enhancements in serum IgG and IgA were seenwhen mice were immunized with Hemophilus Influenza type b polysaccharideconjugate vaccine, Hib coupled to Dt(HibCV) and received 1,25(OH)₂ D₃ onDay 5. Similar results were obtained with topical administration of1,25(OH)₂ D₃ to other antigens, as shown in the following Example.

EXAMPLE 15 Administration of 1,25(OH)₂ D₃ Enhances Antibody Response toVaccinations with Various Antigens

Sex- and age-matched mice [CF-1] were immunized subcutaneously with thefollowing antigens in alum (273 μg/ml):

Chlamydia trachomatus peptide (5 μg)

Hemophilus Influenza untypeable (1.0 μg)

Hemophilus Influenza Type b conjugate vaccine coupled to Dt (500 ng ofHib polysaccharide chemically coupled to 1.25 μg Dr)

Respiratory syncytial virus peptide (1 μg)

Hepatitis B Surface Antigen (1 μg)

HIV gp 120 (0.5 μg)

Neisseria gonorhaeae pilin protein (1 μg)

Diptheria toxoid (1 μg)

One group of mice was administered 2 μg 1,25(OH)₂ D₃ epicutaneously atthe same site on Day 0. Untreated mice were administered the ethanolvehicle without 1,25(OH)₂ D₃. Serum samples and mucosal samples (vaginallavage samples (75 μl of physiological saline)) were collected fromindividual mice at multiple times during the primary response.Individual serum and mucosal samples were evaluated by quantitativeELISA as described in Example 14, using the appropriate antigens. Themean primary antibody responses at Day 28 are shown in Table 3 for theserum (systemic or humoral) antibodies and in Table 4 for the mucosalantibodies. The results show that the serum and mucosal antibodyresponses were enhanced in mice with topical administration of 1,25(OH)₂D₃. Mucosal antibodies (both IgG and IgA) were also detected in othermucosal secretions, including lacrimal, rectal, oral and lung. Similarresults are obtained when all trans-retinoic acid is used in place of1,25(OH)₂ D₃.

                  TABLE 3                                                         ______________________________________                                        Secretory Antibody Production                                                              Systemic Ig (ng/ml)                                                                      Vaccine With                                                       Vaccine    Topical                                                            Only       1,25(OH).sub.2 D.sub.3                                             IgG    IgA     IgG     IgA                                       ______________________________________                                        Chlamydia trachomatus                                                                        <0.02    43      55    85                                      peptide (5 μg)                                                             Hemophilus Influenza                                                                         3235     69      4712  93.2                                    untypeable                                                                    Hemophilus Influenza                                                          type b-CV:                                                                    Hib polysaccharide                                                                           25       12.5    125   76.1                                    (500 ng)                                                                      Diphtheria toxoid                                                                            853      11.6    1285  20.3                                    (1.25 μg)                                                                  Respiratory Syncytial                                                                        2746     225     8440  754                                     virus peptide (1 μg)                                                       Hepatitis B surface                                                                          254      58      902   149                                     antigen (1 μg)                                                             HIV gp120 (.5 μg)                                                                         1356     854     2459  1408                                    Neisseria gonorhoeae                                                                         844      16.3    1841  29.1                                    pilin protein (1 μg)                                                       Diphtheria toxoid (1 μg)                                                                  1233     23      1640  137                                     ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Secretory Antibody Production                                                               Mucosal Ig (pg/ml)                                                                      Vaccine With                                                        Vaccine   Topical                                                             Only      1,25(OH).sub.2 D.sub.3                                              IgG   IgA     IgG      IgA                                      ______________________________________                                        Chlamydia trachomatus                                                                         <20     397     759    2234                                   peptide (5 μg)                                                             Hemophilus Influenza                                                                          719     1384    2081   1865                                   untypeable                                                                    Hemophilus Influenza                                                          type b-CV:                                                                    Hib polysaccharide                                                                            180     440     280    1720                                   (500 ng)                                                                      Diphtheria toxoid                                                                             310     400     590    1400                                   (1.25 μg)                                                                  Respiratory Syncytial                                                                         <20     <20     1544   1264                                   virus peptide (1 μg)                                                       Hepatitis B surface                                                                           <20     <20     450    250                                    antigen (1 μg)                                                             HIV gp120 (.5 μg)                                                                          35      45      1428   755                                    Neisseria gonorhoeae                                                                          6341    1063    10235  5486                                   pilin protein (1 μg)                                                       Diphtheria toxoid (1 μg)                                                                   <20     60      1.8    1125                                   ______________________________________                                    

The above example was repeated, using 0.1 μg 1,25(OH)₂ D₃ in the vaccine(in a total volume of 25 μl with alum (250 μg/ml)) instead of topicaladministration of the 1,25(OH)₂ D₃. Identical results were obtained asset forth in Tables 3 and 4.

EXAMPLE 16 Comparative Effect of 1,25(OH)₂ D₃ and All Trans-RetinoicAcid on Immunoalobulin Production

Groups of sex- and age-matched mice [CF-1], 17-24 weeks of age, wereimmunized with 1.0 μg HBSAg in 25 μl alum. The mice in each group wereimmunized with either vaccine alone, vaccine with 0.1 μg 1,25(OH)₂ D₃,or vaccine with 5 μl all trans-retinoic acid. The agents wereincorporated directly into the vaccine mixture. Individual serum(systemic) samples and mucosal samples (vaginal lavage samples (75 μl ofphysiological saline)) were collected at weekly intervals during theprimary response. The mean quantities of antibodies (IgG and IgA)detected in the serum and mucosal secretions 28 days after a singleimmunization are shown in Table 5. The results show that both 1,25(OH)₂D₃ and all trans-retinoic acid enhance both the serum and mucosalantibody response.

                  TABLE 5                                                         ______________________________________                                        Antibody Production with 1,25(OH).sub.2 D.sub.3                               or All Trans-Retinoic Acid                                                                    Systemic Ig      Mucosal Ig                                   Composition     (ng/ml)          (ng/ml)                                      of Vaccine      IgG    IgA       IgG  IgA                                     ______________________________________                                        Vaccine only    225    160       35   <20                                     Vaccine w/ 0.1 μg                                                                          457    494       352  341                                     1,25(OH).sub.2 D.sub.3                                                        Vaccine w/ 5 μg All                                                                        295    531       437  311                                     Trans-Retinoic Acid                                                           ______________________________________                                    

EXAMPLE 17 Administration of DHEA and 1,25(OH)₂ D₃ in Vaccine EnhancesSerum and Mucosal Antibody Response

Groups of five mature adult C3H mice were immunized with 0.1 μginactivated Influenza-A Beijing strain in 25 μl of alum (273 μg/ml) inthe hind footpad. The mice in each group were immunized with eithervaccine alone, vaccine plus 2 μg DHEA, vaccine plus 0.1 μg 1,25(OH)₂ D₃or vaccine with both 2 μg DHEA and 0.1 μg 1,25(OH)₂ D₃. The agents wereincorporated directly into the vaccine mixture. Individual serum(systemic) samples and mucosal samples (vaginal lavages (75 μlphysiological saline)) were collected at weekly intervals during theprimary response and evaluated by quantitative ELISA. FIGS. 13A, 13B,13C and 13D show the mean quantities of antibody detected in serum andmucosal secretions 28 days after a single immunization, respectively.The results show that coadministration of DHEA and 1,25(OH)₂ D₃ in thevaccine synergistically enhances both the serum and mucosal antibodyresponse.

EXAMPLE 18 Administration of DHEA and 1,25(OH)₂ D₃ in Vaccine EnhancesSerum and Mucosal Antibody Response

Groups of five mature adult CF1 mice were immunized with 1.0 μg rHBSAgin 25 μl of alum (273 μg/ml) in the hind footpad. The mice in each groupwere immunized with either vaccine alone, vaccine plus 2 μg DHEA,vaccine plus 0.1 μg of 1,25(OH)₂ D₃, or vaccine with both 2 μg DHEA and0.1 μg 1,25(OH)₂ D₃. The agents were incorporated directly into thevaccine mixture. Individual serum (systemic) samples and mucosal samples(vaginal lavages (75 μl of physiological saline)) were collected atweekly intervals during the primary response. FIGS. 14A, 14B, 14C and14D show the mean quantities of antibody detected in serum and mucosalsecretions 21 days after a single immunization. The results show thatcoadministration of DHEA and 1,25(OH)₂ D₃ in the vaccine synergisticallyenhances both the serum and mucosal antibody response.

EXAMPLE 19 Administration of DHEA and All Trans-Retinoic Acid in VaccineEnhances Serum and Mucosal Antibody Response

Groups of five mature adult CF1 mice were immunized with 1.0 μg rHBSAgin 25 μl of alum (273 μg/ml) in the hind footpad. The mice in each groupwere immunized with either vaccine alone, vaccine plus 5.0 μg of alltrans-retinoic acid, or vaccine with both 2 μg DHEA and 5.0 μg alltrans-retinoic acid. The agents were incorporated directly into thevaccine mixture. Individual serum (systemic) samples and mucosal samples(vaginal lavages (75 μl of physiological saline)) were collected atweekly intervals during the primary response. FIGS. 15A, 15B, 15C and15D show the mean quantities of antibody detected in serum and mucosalsecretions 21 days after a single immunization. The results show thatcoadministration of DHEA and all trans-retinoic acid in the vaccinesynergistically enhances both the serum and mucosal antibody response.

While the invention has been disclosed in this patent application byreference to the details of preferred embodiments of the invention, itis to be understood that the disclosure is intended in an illustrativerather than in a limiting sense, as it is contemplated thatmodifications will readily occur to those skilled in the art, within thespirit of the invention and the scope of the appended claims.

What is claimed is:
 1. A method for enhancing an antigen-specificcirculating antibody immune response which comprises administering to anindividual a vaccine and an effective amount of an immunomodulator as avaccine adjuvant, said immunomodulator selected from the groupconsisting of a lymphoid organ modifying agent and a mixture of allimmune response augmenting agent and said lymphoid organ modifyingagent, said immune response augmenting agent having the formula ##STR3##wherein R¹ is ═O;R² is H or halogen; R³ is H with a 5-6 double bond; R⁴is OR⁵ ; R⁵ is H, SO₂ OM, or PO₂ OM; ##STR4## R⁶ and R⁷ may be the sameor different and may be a straight or branched C₁₋₁₄ alkyl, and saidlymphoid organ modifying agent is selected from the group consisting of1,25-dihydroxy Vitamin D₃ and all trans-retinoic acid.
 2. The method ofclaim 1 wherein the immunomodulator is administered up to three hoursprior to administration of said vaccine.
 3. The method of claim 2wherein said vaccine comprises an antigen which is capable of elicitingan immune response against viral hepatitis, influenza, diphtheria,tetanus, pertussis, measles, mumps, rubella, polio, pneumococcus,herpes, respiratory syncytial virus, haemophilus influenza type b,varicella-zoster virus or rabies.
 4. The method of claim 1 wherein theimmunomodulator and vaccine are administered contemporaneously.
 5. Themethod of claim 4 wherein said vaccine comprises an antigen which iscapable of eliciting an immune response against viral hepatitis,influenza, diphtheria, tetanus, pertussis, measles, mumps, rubella,polio, pneumococcus, herpes, respiratory syncytial virus, haemophilusinfluenza type b, varicella-zoster virus or rabies.
 6. The method ofclaim 1 wherein the immunomodulator is administered in a vaccine.
 7. Themethod of claim 6 wherein said vaccine comprises an antigen which iscapable of eliciting an immune response against viral hepatitis,influenza, diphtheria, tetanus, pertussis, measles, mumps, rubella,polio, pneumococcus, herpes, respiratory syncytial virus, haemophilusinfluenza type b, varicella-zoster virus or rabies.
 8. The method ofclaim 1 wherein wherein said immunomodulator is a lymphoid organmodifying agent.
 9. The method of claim 8 wherein said lymphoid organmodifying agent is 1,25-dihydroxy Vitamin D₃.
 10. The method of claim 8wherein said lymphoid organ modifying agent is all trans-retinoic acid.11. The method of claim 8 wherein the amount of said lymphoid organmodifying agent is 0.1-500 μg.
 12. The method of claim 1 wherein whereinsaid immunomodulator is said mixture of an immune response augmentingagent and a lymphoid organ modifying agent.
 13. The method of claim 12,R¹ is ═O, R² is H, R³ is H with a 5-6 double bond, and R⁴ is OH.
 14. Themethod of claim 13 wherein said lymphoid organ modifying agent is1,25-dihydroxy Vitamin D₃.
 15. The method of claim 13 wherein saidlymphoid organ modifying agent is all trans-retinoic acid.
 16. Themethod of claim 12, R¹ is ═OH, R² is H, R³ is H with a 5-6 double bond,and R⁴ is OH.
 17. The method of claim 16 wherein said lymphoid organmodifying agent is 1,25-dihydroxy Vitamin D₃.
 18. The method of claim 16wherein said lymphoid organ modifying agent is all trans-retinoic acid.19. The method of claim 12, R¹ is ═O, R² is H, R³ is H with a 5-6 doublebond, and R⁴ is OH.
 20. The method of claim 19 wherein said lymphoidorgan modifying agent is 1,25-dihydroxy Vitamin D₃.
 21. The method ofclaim 19 wherein said lymphoid organ modifying agent is alltrans-retinoic acid.
 22. The method of claim 12, R¹ is ═O, R² is H, R³is H with a 5-6 double bond, and R⁴ is OH.
 23. The method of claim 22wherein said lymphoid organ modifying agent is 1,25-dihydroxy VitaminD₃.
 24. The method of claim 22 wherein said lymphoid organ modifyingagent is all trans-retinoic acid.
 25. The method of claim 12 whereinsaid lymphoid organ modifying agent is 1,25-dihydroxy Vitamin D₃. 26.The method of claim 12 wherein said lymphoid organ modifying agent isall trans-retinoic acid.
 27. The method of claim 12 wherein the amountof said immune response augmenting agent is 0.1-1,000 μg.
 28. The methodof claim 27 wherein the amount of said lymphoid organ modifying agent is0.1-500 μg.
 29. The method of claim 12 wherein the amount of saidlymphoid organ modifying agent is 0.1-500 μg.
 30. The method of claim 1wherein said immunomodulator is administered epicutaneously.
 31. Themethod of claim 1 wherein said immunomodulator is administeredintramuscularly.
 32. The method of claim 1 wherein said immunomodulatoris administered intradermally.
 33. The method of claim 1 wherein saidimmunomodulator is administered subcutaneously.
 34. The method of claim1 wherein the immunomodulator is administered up to three hoursfollowing administration of the vaccine.
 35. The method of claim 34wherein said vaccine comprises an antigen which is capable of elicitingan immune response against viral hepatitis, influenza, diphtheria,tetanus, pertussis, measles, mumps, rubella, polio, pneumococcus,herpes, respiratory syncytial virus, haemophilus influenza type b,varicella-zoster virus or rabies.
 36. The method of claim 1 wherein saidimmunomodulator is administered topically.